Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

The localization,metabolism and biological activity of gibberellins in maturing and germinating seeds of Pisum sativum cv. Progress No. 9

Gibberellin A₂₀ (GA₂₀), GA₂₉ and GA₂₉-catabolite were quantified in cotyledons, embryonic axes, and testas of Pisum sativum cv. Progress No. 9 throughout the final stages of seed maturation and during germination. Stable isotope-labelled GAs were used as internal standards in conjunction with combined gas chromatography-mass spectrometry. Gibberellin A₂₀ and GA₂₉ were mainly located in the cotyledons of maturing seeds, and GA₂₉-catabolite was predominantly located in the testa. Stable isotope- and radio-labelled GA₂₀ and GA₂₉ were fed to both intact seeds developing in vivo, and to isolated seed parts cultured in vitro. The combined results of in-vivo and in-vitro feeds indicated that GA₂₀ is metabolised to GA₂₉ in the cotyledons, that GA₂₉ is transported from the cotyledons to the testa, and that GA₂₉ is metabolised to GA₂₉-catabolite in the testa. Although the metabolism of GA₂₀ in the cotyledons and of GA₂₉ in the testa has been shown definitively, the mobility of GA₂₉ has not yet been demonstrated directly. During seed desiccation and germination GA₂₉-catabolite and products arising from it are transferred from the testa into the embryo. There is no evidence of a physiological function for GA₂₉-catabolite in germination or early seedling growth. Use of a growth retardant indicates that seedling growth, but not germination, is dependent on de-novo GA biosynthesis.     

Gibberellin A20 in seed of Pisum sativum L., cv. Alaska

Summary The main gibberellin in immature seed of Pisum sativum L., cv. Alaska, is identified as GA₂₀ by GC-MS. GA₉ may also be present.     

Identification,quantitation and distribution of gibberellins in fruits of Pisum sativum L. cv. Alaska during pod development

In addition to the previously-reported gibberellins: GA₁; GA₈, GA₂₀ and GA₂₉ (García-Martínez et al., 1987, Planta 170, 130–137), GA₃ and GA₁₉ were identified by combined gas chromatography-mass spectrometry in pods and ovules of 4-d-old pollinated pea (Pisum sativum cv. Alaska) ovaries. Pods contained additionally GA₁₇, GA₈₁ (2-hydroxy GA₂₀) and GA₂₉-catabolite. The concentrations of GA₁, GA₃, GA₈, GA₁₉, GA₂₀ and GA₂₉ were higher in the ovules than in the pod, although, with the exception of GA₃, the total content of these GAs in the pod exceeded that in the seeds. About 80% of the GA₃ content of the ovary was present in the seeds. The concentrations of GA₁₉ and GA₂₀ in pollinated ovaries remained fairly constant for the first 12 ds after an thesis, after which they increased sharply. In contrast, GA₁ and GA₃ concentrations were maximal at 7 d and 4–6 d, respectively, after anthesis, at about the time of maximum pod growth rate, and declined thereafter. Emasculated ovaries at anthesis contained GA₈, GA₁₉ and GA₂₀ at concentrations comparable with pollinated fruit, but they decreased rapidly. Gibberellins a₁ and A₃ were present in only trace amounts in emasculated ovaries at any stage. Parthenocarpic fruit, produced by decapitating plants immediately above an emasculated flower, or by treating such flowers with 2,4-dichlorophenoxyacetic acid or GA₇, contained GA₁₉ and GA₂₀ at similar concentrations to seeded fruit, but very low amounts of GA₁ and GA₃ Thus, it appears that the presence of fertilised ovules is necessary for the synthesis of these last two GAs. Mature leaves and leaf diffusates contained GA₁, GA₈, GA₁₉ and GA₂₀ as determined by combined gas chromatography-mass spectrometry using selected ion monitoring. This provides further evidence that vegetative tissues are a possible alternative source of GAs for fruit-set, particularly in decapitated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - KRI Kovats retention index - m/z mass to charge ratio We thank Mr M.J. Lewis for qualitative GC-MS analyses and Ms M.V. Cuthbert (LARS), R. Martinez Pardo and T. Sabater (IATA) for technical assistance. We are also grateful to Professor B.O. Phinney, University of California, Los Angeles, for gifts of [17-¹³C]GA₈ and -GA₂₉ and to Mr Paul Gaskin, University of Bristol, for the mass spectrum of GA₂₉-catabolite and for a sample of GA₈₁ The work in Spain was supported by Dirección General de Investigación Cientifica y Técnica (grant PB87-0402 to J.L.G.-M.). We also acknowledge the British Council and Ministerio de Educacion y Ciencia for travel grants through Accion Integrada Hispano-Britanica 56/142 (J.L.G.-M. and P.H.).     

Characterization of abscisic acid in chloroplasts of Pisum sativum L. cv. Alaska by combined gas-chromatography-mass spectrometry

Summary Abscisic acid was characterized from extracts of isolated pea chloroplasts by combined gas-chromatography-mass spectrometry. By single ion monitoring of the base peak (m/e 190) in the mass spectrum, 8.6 g ABA were detected in chloroplasts isolated from 1 kg fresh weight of pea shoots.GC-MS combined gas chromatography-mass spectrometry     

Metabolism of [13C1]gibberellin A29 to [13C1]gibberellin-catabolite in maturing seeds of Pisum sativum cv. Progress No. 9

The metabolism of GA₂₉ during seed maturation in Pisum sativum cv. Progress No. 9 was further investigated. [17-¹³C₁]GA₂₉ was metabolised to a GA-catabolite (structure 3), with incorporation of the [¹³C] label from the GA₂₉ substrate into the GA-catabolite being demonstrated by GC-MS. Quantitation of the GA-catabolite using GC-MS was achieved by adding GA-catabolite, labelled with [¹⁸O], to seed extracts as an internal standard. At least 50% conversion of [¹³C₁]GA₂₉ to [¹³C₁]GA-catabolite was demonstrated with the build up of exogenous [¹³C₁]GA-catabolite strictly paralleling the accumulation of native GA-catabolite. These results strongly suggest that conversion of GA₂₉ to the GA-catabolite is a natural metabolic step occurring during the final stages of seed maturation. 25 g per seed of native GA-catabolite was recorded in 37 day old seeds. Some problems encountered in the analysis of extracts containing the GA-catabolite are discussed briefly.Abbreviations BSTFA bis(trifluoromethylsilyl)acetamide - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - Me methyl ester - SICM selected ion current monitoring - TMSi trimethylsilyl ether     

Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA₃-equivalents) and 12–13 (270 g GA₃-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA₁, GA₃, GA₄ and GA₈ were active in the Phaseolus bioassay but GA₈-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA₄, GA₁₉ and GA₁. The identity of GA₄ in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA₁₉ and GA₁ were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727.     

Organization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea (Pisum sativum L. cv Progress No. 9) leaves

The organization of the enzymes of de novo pyrimidine nucleotide biosynthesis in pea (Pisum sativum L. cv Progress No. 9) has been studied. The first three enzymes of the pathway, carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase, are readily separable from one another; they are not part of a multifunctional complex. The final two activities of the pathway, orotate phosphoribosyltransferase and orotidylate decarboxylase, copurify and appear to be complexed in vivo. This organizational pattern is distinct from those reported for bacteria, yeast, and mammals. The differences in organization, in a pathway which is present in all organisms, make the pyrimidine biosynthetic pathway a very interesting candidate for evolutionary studies.     

Identification of gibberellin A20, abscisic acid,and phaseic acid from flowering Bryophyllum daigremontianum by combined gas chromatography-mass spectrometry

Summary The presence of abscisic and phaseic acid in a purified acidic extract from flowering plants of the long-short-day plant Bryophyllum daigremontianum [(R. Hamet and Perr.) Berg.] was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of their methyl esters. Gibberellin A₂₀ (GA₂₀) was identified by GC-MS of the methyl ester and the trimethylsilyl ether of the methyl ester. The following levels of the 3 compounds per kg fresh weight were estimated: Abscisic acid, 5.5 g; phaseic acid, 9.4g; gibberellin A₂₀, 0.8 g. When GA₂₀ and four other GAs were applied to Bryophyllum under shortday conditions, the order of effectiveness for induction of flower formation was: GA₂>GA₁>GA₅=GA₇>GA₂₀. The low biological activity of the native GA₂₀ is discussed.     

Detection and identification of gibberellins in needles of silver fir (Abies alba Mill.) by combined gas chromatography-mass spectrometry

Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-year old (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected by the Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA₉ was present in small amounts, while a major component was a cellulase-hydrolysable GA₉ conjugate which was assumed to be GA₉ glucosyl ester. It is proposed that GA₉ glucosyl ester plays a key role in the regulation of endogenous GA levels in silver fir needles.     

Metabolism of gibberellin A29 in seeds of Pisum sativum cv. Progress No. 9; Use of [2H] and [3H]GAs,and the identification of a new GA catabolite

The metabolism of GA₂₉ in maturing seeds of Pisum sativum cv. Progress No. 9 was further investigated, and the utility of ²H-labelled GAs in conjuction with GC-MS is illustrated. Using [2-²H₁]GA₂₉ as an internal standard, endogenous GA₂₉ was shown to reach a maximal level (ca. 10 g/seed) 27 days from anthesis, and to decline to ca. 1.6 g/seed in mature seeds. In a time-course feed the metabolism of [2-²H₁] [2-³H₁]GA₂₉ applied to 27 day old seeds, and of endogenous GA₂₉, was compared from the ¹H:²H ratios in the recovered GA₂₉. Although both [2-²H₁] [2-³H₁]GA₂₉ and endogenous GA₂₉ were metabolised to the same limited extent to a putative conjugate, in the main metabolic process endogenous GA₂₉ was preferentially converted to an untraceable (i.e. unlabelled) metabolite. In contrast, endogenous GA₂₉ and [1,3-²H₂] [1,3-³H₂]GA₂₉, derived from [1,3-²H₂] [1,3-³H₂]GA₂₀ in a time-course feed, were metabolised in an identical manner. In the latter case isotope loss precluded identification of the metabolite. The structure (8) has been assigned to a GA catabolite present in maturing seeds and seedlings of pea. The isotope data are consistent with this compound being the hitherto untraced metabolite of GA₂₉ in pea.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - M⁺ molecular ion - Me methyl ester - R_T retention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethylsilyl ether     

Identification of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry

A method for the monitoring of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids was developed, involving GC-MS with multiple selected-ion monitoring. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after having been concentrated to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed, which showed good behaviour in GC-MS with electron impact ionization. Its molecular ion, m/z 609, and its main fragments, m/z 576, 534, 522 and 508, were selected as the ions for identification owing to their relative peak intensities and characteristics. The target drug was identified based on its retention time, its selected multiple ions and their relative intensities. This method was successfully used for the detection of dihydroetorphine in blood and urine from a dihydroetorphine addict and a poisoned patient, respectively.     

Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

Identification of 6-hydroxymelatonin in normal human urine by gas chromatography-mass spectrometry.

The major melatonin metabolite, 6-hydroxymelatonin, has been identified in normal human urine by gas chromatography-mass spectrometry (gc-ms) after rapid acid hydrolysis, extraction, and derivatization. An estimate of the amount detected (approximately 20 ng/ml) is consistent with calculated rates of melatonin synthesis and turnover.     

Identification of glycation at the N-terminus of albumin by gas chromatography-mass spectrometry.

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization

In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight. Excess ROS including singlet oxygen (¹O₂) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ), a generator of O₂ ^·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound ¹O₂ was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position. In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including ¹O₂ plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor for crop yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of benzophenanthridine alkaloids from Bocconia arborea by gas chromatography-mass spectrometry

A methanol extract of the bark of Bocconia arborea was fractionated on silica gel and the fractions analysed using gas chromatography coupled with mass spectrometry (GC-MS). Several benzophenanthridine alkaloids were identified including dihydrosanguinarine, oxysanguinarine, 11-acetonyldihydrochelerythrine, dihydrochelerythrine, chelerythrine, chelerythridimerine and angoline as the principal constituents. The results show that the direct GC-MS analysis of these alkaloids is possible with a clear distinction between the compounds. The technique is shown to be a valuable tool and an alternative technique to classical phytochemical procedures permitting the fast analysis of alkaloids mixtures.